Ergothioneine is a non-essential L-amino acid found naturally in the body. It is a very strong antioxidant, and because it is a carnitine analog, it may also have activity in aerobic ATP production. It is synthesized by types of fungi but not mammals, who must acquire it in their diet. They do so by direct consumption of fungi such as mushrooms, or from grains which have themselves taken up ergothioneine from fungi in their roots. Methods for the laboratory preparation of synthetic ergothioneine have been described in, for example, H. Heath, A. Lawson and C. Rimington. “2-Mercaptoglyoxalanes. Part I. The synthesis of Ergothioneine”, J. Chem Soc. (1951) pp 2215-2217 and in U.S. Pat. No. 5,438,151.
It has been observed that ergothioneine is quite stable at acidic pH. At pH 5 a 0.05% solution remains within 1% of its concentration after heating 40 C for 60 days. However, the reports of its stability in alkali are mixed in the literature. See, for example, Philip Hartman. “Ergothioneine as antioxidant”, Methods Enzymol. 186:310-318, 1990 at p. 311 (“ . . . the sulfur atom of Ergothioneine is remarkably stable to alkali”); H. Heath and G. Toennies. “The preparation and properties of Ergothioneine disulphide”, J. Chem. Soc. pages 204-210, (1958) at p. 204 (“the sulphur is completely unaffected by boiling 50% aqueous potassium hydroxide.”); Oxis International, Inc. Compound Monograph “L-Ergothioneine. Revision III”, at p. 4 (“at physiological pH LE [L-Ergothioneine] does not auto-oxidize and is therefore very stable in aqueous solution.” . . . “remarkably stable to strong alkali”); and Jinzhu Xu and Jean Claude Yadan. “Synthesis of L-(+)-Ergothioneine”, J. Org. Chem. 60:6296-6301, 1995 at p. 6296 (“this explains its stability toward oxidative dimerization”).
On the other hand, see U.S. Pat. No. 5,438,151, at column 1, line 40 (“the very ready β-elimination of the trimethylammonium group in alkaline medium”); Donald Melville. “Ergothioneine. Vitamins and Hormones”, 17:155-204, 1959, at p. 161 (“When Ergothioneine was boiled with 50% KOH solution, trimethylamine was evolved and a yellow acid C6H6O2N2S was formed”), and at p. 165 (“The two most characteristic chemical reactions of Ergothioneine are the ready oxidation of the sulfur atom and the lability of the trimethylammonium radical toward alkali . . . the formation of thiolurocanic acid and trimethylamine by the treatment of Ergothioneine with hot, concentrated alkali has already been considered.”).
Ergothioneine and its solutions have no odor, despite the presence of sulfur, because the sulfur is in the thione conformation (C═S), which has no odor, rather than the sulfhydryl conformation (C—SH), which has the odor of rotten eggs. The literature does not mention the appearance of any odor during storage under any conditions. In fact, European Patent No. EP0483426 describes ergothioneine as one of the preferred ingredients in a deodorant composition for topical application to the skin. The contents of the documents cited herein are incorporated herein by reference in their entirety.
The inventor has observed that a cosmetic formulation containing 0.3% ergothioneine at pH 4.2-5.0, stored over several months at room temperature, when rubbed onto a person's skin, presented a fishy, amine odor to that person. Other cosmetic formulations with more dilute solutions of 0.001% or 0.0005% at pH 7 also produced a fishy, amine odor when rubbed onto a person's skin. However, in the cases of the more dilute solutions, the odor was detected by some individuals and not others. The odor was produced by stored samples which retained the same concentration of the ergothioneine as they did initially.
The fishy, amine odor associated with ergothioneine is a disadvantage in commercialization, particularly in oral pharmaceuticals, injectibles pharmaceuticals, topical pharmaceuticals, cosmetic products, nutritional supplements, nutritional drinks, and other consumer products.
Those skilled in the art have found the removal or prevention of the fishy, amine odor in aqueous solutions to be a particularly difficult problem. U.S. Pat. No. 5,814,233 describes a method for remediating methyl amine odors in aqueous systems using a compound with amide or imide functionality, such as a hydantoin composition, in the presence of hydrogen peroxide. The patent further proposes reacting choline with hydantoin and hydrogen peroxide. In U.S. Pat. Nos. 5,137,982 and 5,078,913, the inventors describe a method for removing odor from a solution containing trimethylamine and choline chloride using polybasic acid to react with the choline chloride. The acids described include strong acids such as sulfuric acid. U.S. Pat. No. 4,845,289 describes a method for the removal or reduction of odor by use of methyl chloride at temperatures above 50° C. As disclosed in the patent, “In these products as well as others, there frequently remains upon completion of the reaction excess trimethylamine which is odiferous and has undesirable toxicological problems. The residual trimethylamine is difficult to remove completely from aqueous system because of its high solubility in water, and extensive purging with an inert gas and/or removal of water is necessary to also remove substantial quantities of amine. This can be a time-consuming and energy intensive process.”
In addressing this concern, the initial assumption of the inventor was that ergothioneine dimerizes by forming a disulfide link between two molecules over time, which was not detected by the analytical method. The inventor then reasoned that the dimer reacts on the surface of a person's skin to release a compound that produces an odor. The most widely recognized component of the human body which can produce a fishy, amine smell is choline, which is a component of prevalent skin lipids such as phosphatidyl choline. Persons with large amounts of choline in their diets, or who are defective in metabolizing choline, develop a fishy, amine odor on their breaths and their bodies. It was therefore hypothesized by the inventor that the reactant, perhaps choline, differed from one person's skin to another, which would explain why some people detected the odor after rubbing it on their skin and others did not. In addition, it was theorized that the reaction required a few seconds of heat from the skin, which would explain why the odor appeared in a delayed fashion.
However, the inventor has now appreciated that the source of the odor associated with the manufacture and storage of ergothioneine is not related to the presence of choline, and that none of the methods of the prior art are useful in preventing the formation of the odor, produced after application to human skin, of a product that contains ergothioneine but that does not contain choline chloride.
The present invention describes practical methods which have now been discovered to prevent the formation of the odor and/or eliminate the odor, and to thereby remove this hindrance to commercialization.
Without being limited to one particular theory, it is believed that the odor associated with ergothioneine may be caused by the formation of minute quantities of trimethylamine in solution at neutral or even acidic pH over time. Trimethylamine is a gas which has a strong ammonium odor but in trace amounts has a fishy, amine odor. It is produced by rotting fish and other decaying foods. The human nose is sensitive to minute quantities of trimethylamine (odor threshold 25 parts per billion), which probably evolved as a defense mechanism to detect contaminated foods and thus to avoid illness. The gas is formed within the aqueous phase of ergothioneine mixtures and remains dissolved, thus masking the odor when the mixture is smelled directly. However, when the ergothioneine mixture is applied to skin, the aqueous components of the mixture are absorbed and the heat of the skin volatilizes the trimethylamine. This may explain why, after rubbing the mixture onto skin, the odor is detectable only after a few seconds. Therefore, it is theorized that the variation in perception of the odor by people is not a result of differences in their skins or choline content but rather because of the significant differences in olfactory sensitivity in the human population, i.e. the ability to detect trimethylamine in trace amounts. In fact, 5-7% of the human population is unable to smell it at all, a condition called anosmia. HPLC analyses of the ergothioneine concentrations did not detect the reduction in ergothioneine or the formation of trimethylamine because the changes were in trace amounts below the limits of detection of the HPLC, but not below the limit of the human nose to detect trimethylamine.
This observation by the inventor was confirmed by reproducing the fishy, amine odor by boiling 2 mM (0.046%) ergothioneine with 0.1 N NaOH (pH 11.5) for 10 minutes. The odor was not ammonia-like, and was detectable by all persons. This became the assay system used to test agents and procedures for reduction or prevention of the odor.
It now has been discovered by the inventor that certain trimethylamine absorbers, which also may be referred to as binders or conjugators, are able both to prevent the formation of trimethylamine from ergothioneine and to bind to trimethylamine after its formation to prevent it from escaping as a gas detectable by the nose.
In one embodiment of the present invention, the trimethylamine binder is an acid, especially a weak acid.
In a further embodiment of the present invention, the trimethylamine binder is sulfur dioxide or a compound which generates sulfur dioxide upon its dissolution in water (i.e. sulfur dioxide donors). The sulfur dioxide is able to bind trimethylamine after its formation to prevent it from escaping as a gas detectable by the nose. Thus, sulfur dioxide can function like the conjugated base of a weak acid to prevent or reduce the odor of ergothioneine during storage.